Mitochondria in pathophysiology

Figure 1. Protective effect of CsA on liver damage caused by in vivo treatment with LPS plus D-GalN. Paraffin-embedded liver sections were stained with hematoxylin-eosin (a,b) or processed for the TUNEL reaction (a',b') 8 h after treatment with LPS plus D-GalN. Rats had been pretreated with either vehicle (a,a') or with CsA (b,b').

Figure 1. Protective effect of CsA on liver damage caused by in vivo treatment with LPS plus D-GalN. Paraffin-embedded liver sections were stained with hematoxylin-eosin (a,b) or processed for the TUNEL reaction (a',b') 8 h after treatment with LPS plus D-GalN. Rats had been pretreated with either vehicle (a,a') or with CsA (b,b').
[click image to enlarge]

Mitochondria are central to the life of eukaryotic cells. In recent years, it has become clear that mitochondria also play a key role in the pathways to cell death. This role of mitochondria cannot be explained by a mere "loss of function" resulting in an energetic deficit. Rather, it is increasingly recognized as an "active" process that is intimately linked to intracellular signalling, and is mediated by regulated effector mechanisms that come into play in a wide variety of conditions. A large body of observations from the fields of Immunology, Toxicology, Oncology, Neurology and Cardiology, to name a few, testifies about the centrality of this issue. The involvement of mitochondria in cell death has been investigated for many years, particularly in relation to Ca²⁺ homeostasis.

Further impulse to mitochondrial studies in the context of cell death came with the identification of mitochondrial proteins that participate in modulating the execution phase of apoptosis. These proteins can be grouped in two classes: (i) pro- and anti-apoptotic members of the Bcl-2 family that largely localize to the outer mitochondrial membrane; (ii) proteins that are released during apoptosis to activate caspase-dependent (cytochrome c, smac-diablo) and caspase-independent cell death (apoptosis inducing factor, endonuclease G). One of the key issues is the mechanism through which these proteins involved in execution of the cell death program are released from mitochondria. My laboratory is studying this problem with specific emphasis on the potential role of the permeability transition pore (PTP), an inner membrane, high conductance channel inhibited by cyclosporin A (CsA). The PTP is currently one of the most appealing molecular targets in the mitochondrial execution of the death program, and a point of regulation where relevant signalling pathways may converge. Indeed, pore opening leads to (i) energy dissipation because it causes depolarization, with ensuing ATP depletion which is largely contributed by hydrolysis by the mitochondrial F1Fo ATPase; (ii) increase of cytosolic [Ca²⁺], caused by lack of mitochondrial uptake and/or by mitochondrial release coupled to decrease of the ATP-dependent activity of the organellar and plasma membrane Ca²⁺ pumps; and possibly (iii) release of apoptogenic proteins as a result of mitochondrial swelling that leads to outer membrane rupture. Our laboratory is at the forefront of research in the study of the regulation of the PTP in a variety of disease models in vitro as well as in vivo.

Research topic 1: Role of cyclophilin D in regulation of the permeability transition.

One of the most interesting features of the PTP is inhibition by CsA, an effect that presumably occurs through cyclophilin (CyP) D, a matrix peptidyl-prolyl-cis-trans-isomerase which would be the endogenous PTP modulator. In collaboration with the Forte Laboratory (Portland, OR) we have generated mice where the Ppif gene encoding for mitochondrial CyP-D had been inactivated. Mitochondria from Ppif^-/- mice had no CyP-D, and displayed a striking desensitization of the PTP to Ca²⁺, in that pore opening required about twice the Ca²⁺ load necessary to open the pore in strain-matched, wild-type mitochondria. Mitochondria lacking CyP-D were insensitive to CsA, which increased the Ca²⁺ retention capacity only in mitochondria from wild-type mice. The PTP response to ubiquinone 0, depolarization, pH, adenine nucleotides and thiol oxidants was similar in mitochondria from wild-type and Ppif^-/- mice. These experiments demonstrate that (i) the PTP can form and open in the absence of CyP-D; (ii) that CyP-D represents the target for PTP inhibition by CsA; and (iii) that CyP-D modulates the sensitivity of the PTP to Ca²⁺, but not its regulation by the proton electrochemical gradient, adenine nucleotides and oxidative stress. These results have major implications for our current understanding of the PTP and its modulation in vitro and in vivo.

Research topic 2: Lipid signalling to mitochondria.

A class of lipid mediators with potential mitochondrial effects is represented by fatty acids. Besides being excellent metabolic substrates, fatty acids are essential factors in the activation of thermogenin (UCP1) in brown fat, but they also uncouple mitochondria from a variety of sources through two mechanisms: (i) a dissipative cycle that involves uptake of the fatty acid anion via the adenine nucleotide carrier and diffusion of the protonated species via the lipid bilayer; and (ii) via opening of the PTP. The relationships between these two events are complex because the PTP is voltage-dependent, and opening can be indirectly triggered by depolarization caused by fatty acid cycling through mechanism (i). We have established that the effects on the PTP are selective for unsaturated fatty acids, and we have establisehd the structure-activity relationship for fatty acids with varying chain length and degree of unsaturation. Specifically, our experiments have shown that arachidonic acid is able to induce PTP opening in isolated mitochondria and intact cells, which results in cell death. Since production of arachidonic acid can be triggered by A23187, we are studying the effects of the divalent cation ionophore A23187 on cellular apoptotic signalling.

Research topic 3: Mitochondria in muscle cell death.

Figure 2. Mitochondrial ultrastructure in muscle cell cultures from healthy donor and UCMD patients. a-e, electron micrographs of myoblasts from healthy donor (a) and UCMD patients (b-d) plated on plastic or on collagen VI (e); insets in a and b are higher magnifications from the same samples; f-h, representative examples of swollen mitochondria found in myoblasts of patients. Bar, 300 nm.

Figure 2. Mitochondrial ultrastructure in muscle cell cultures from healthy donor and UCMD patients. a-e, electron micrographs of myoblasts from healthy donor (a) and UCMD patients (b-d) plated on plastic or on collagen VI (e); insets in a and b are higher magnifications from the same samples; f-h, representative examples of swollen mitochondria found in myoblasts of patients. Bar, 300 nm.
[click image to enlarge]

We have recently shown that the PTP plays an unexpected pathogenetic role in a mouse model of Collagen VI deficiency, which is the cause of Bethlem Myopathy (BM) and Ullrich Congenital Muscular Dystrophy (UCMD) in humans. Treatment with proper doses of CsA allowed us to cure the ultrastructural muscle fiber lesions of mitochondria and sarcoplasmic reticulum in what represents the first example of a successful pharmacological treatment of a genetic model of muscle disease. In order to assess whether mitochondrial dysfunction also plays a role in human Collagen VI deficiencies we have performed an investigation of mitochondrial function in myoblast cultures from UCMD patients. These studies revealed that addition of oligomycin or rotenone to UCMD but not to cultures from control donors caused mitochondrial depolarization. CsA reverted the patient cells' phenotype, suggesting that inappropriate PTP opening may play a role in the pathogenetic mechanism of human Collagen VI myopathies. These results have allowed to start a pilot trial based on treatment of patients with CsA.

Synoptic CV

Appointments

Honours

Selected VIMM Publications

• Baron A, Mancini M, Caldwell E, Cabrelle A, Bernardi P, Pagano F (2009) Serenoa repens extract targets mitochondria and activates the intrinsic apoptotic pathway in human prostate cancer cells. BJU Int. 103:1275-83.
• Cereghetti GM, Stangherlin A, Martins de Brito O, Chang CR, Blackstone C, Bernardi P, Scorrano L (2008) Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria. Proc. Natl. Acad. Sci. U.S.A. 105:15803-8.
• Milanesi E, Costantini P, Gambalunga A, Colonna R, Petronilli V, Cabrelle A, Semenzato G, Cesura AM, Pinard E, Bernardi P (2006) The mitochondrial effects of small organic ligands of BCL-2: sensitization of BCL-2-overexpressing cells to apoptosis by a pyrimidine-2,4,6-trione derivative. J. Biol. Chem. 281:10066-72.
• Penzo D, Petronilli V, Angelin A, Cusan C, Colonna R, Scorrano L, Pagano F, Prato M, Di Lisa F, Bernardi P (2004) Arachidonic acid released by phospholipase A(2) activation triggers Ca²⁺-dependent apoptosis through the mitochondrial pathway. J. Biol. Chem. 279:25219-25.

VIMM Publications

• Baron A, Mancini M, Caldwell E, Cabrelle A, Bernardi P, Pagano F (2009) Serenoa repens extract targets mitochondria and activates the intrinsic apoptotic pathway in human prostate cancer cells. BJU Int. 103:1275-83.
• Cereghetti GM, Stangherlin A, Martins de Brito O, Chang CR, Blackstone C, Bernardi P, Scorrano L (2008) Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria. Proc. Natl. Acad. Sci. U.S.A. 105:15803-8.
• Milanesi E, Costantini P, Gambalunga A, Colonna R, Petronilli V, Cabrelle A, Semenzato G, Cesura AM, Pinard E, Bernardi P (2006) The mitochondrial effects of small organic ligands of BCL-2: sensitization of BCL-2-overexpressing cells to apoptosis by a pyrimidine-2,4,6-trione derivative. J. Biol. Chem. 281:10066-72.
• Soriano ME, Nicolosi L, Bernardi P (2004) Desensitization of the permeability transition pore by cyclosporin a prevents activation of the mitochondrial apoptotic pathway and liver damage by tumor necrosis factor-alpha. J. Biol. Chem. 279:36803-8.
• Penzo D, Petronilli V, Angelin A, Cusan C, Colonna R, Scorrano L, Pagano F, Prato M, Di Lisa F, Bernardi P (2004) Arachidonic acid released by phospholipase A(2) activation triggers Ca²⁺-dependent apoptosis through the mitochondrial pathway. J. Biol. Chem. 279:25219-25.
• Gramaglia D, Gentile A, Battaglia M, Ranzato L, Petronilli V, Fassetta M, Bernardi P, Rasola A (2004) Apoptosis to necrosis switching downstream of apoptosome formation requires inhibition of both glycolysis and oxidative phosphorylation in a BCL-X(L)- and PKB/AKT-independent fashion. Cell Death Differ. 11:342-53.

Additional Publications

• Tiepolo T, Angelin A, Palma E, Sabatelli P, Merlini L, Nicolosi L, Finetti F, Braghetta P, Vuagniaux G, Dumont JM, Baldari CT, Bonaldo P, Bernardi P (2009) The cyclophilin inhibitor Debio 025 normalizes mitochondrial function, muscle apoptosis and ultrastructural defects in Col6a1(-/-) myopathic mice. Br. J. Pharmacol. 157:1045-52.
• Palma E, Tiepolo T, Angelin A, Sabatelli P, Maraldi NM, Basso E, Forte MA, Bernardi P, Bonaldo P (2009) Genetic ablation of cyclophilin D rescues mitochondrial defects and prevents muscle apoptosis in collagen VI myopathic mice. Hum. Mol. Genet. 18:2024-31.
• Basso E, Petronilli V, Forte MA, Bernardi P (2008) Phosphate is essential for inhibition of the mitochondrial permeability transition pore by cyclosporin A and by cyclophilin D ablation. J. Biol. Chem. 283:26307-11.
• Merlini L, Angelin A, Tiepolo T, Braghetta P, Sabatelli P, Zamparelli A, Ferlini A, Maraldi NM, Bonaldo P, Bernardi P (2008) Cyclosporin A corrects mitochondrial dysfunction and muscle apoptosis in patients with collagen VI myopathies. Proc. Natl. Acad. Sci. U.S.A. 105:5225-9.
• Angelin A, Tiepolo T, Sabatelli P, Grumati P, Bergamin N, Golfieri C, Mattioli E, Gualandi F, Ferlini A, Merlini L, Maraldi NM, Bonaldo P, Bernardi P (2007) Mitochondrial dysfunction in the pathogenesis of Ullrich congenital muscular dystrophy and prospective therapy with cyclosporins. Proc. Natl. Acad. Sci. U.S.A. 104:991-6.
• Ferraro P, Nicolosi L, Bernardi P, Reichard P, Bianchi V (2006) Mitochondrial deoxynucleotide pool sizes in mouse liver and evidence for a transport mechanism for thymidine monophosphate. Proc. Natl. Acad. Sci. U.S.A. 103:18586-91.
• Bernardi P, Krauskopf A, Basso E, Petronilli V, Blachly-Dyson E, Blalchy-Dyson E, Di Lisa F, Forte MA (2006) The mitochondrial permeability transition from in vitro artifact to disease target. FEBS J. 273:2077-99.
• Krauskopf A, Eriksson O, Craigen WJ, Forte MA, Bernardi P (2006) Properties of the permeability transition in VDAC1(-/-) mitochondria. Biochim. Biophys. Acta 1757:590-5.
• Giorgio M, Migliaccio E, Orsini F, Paolucci D, Moroni M, Contursi C, Pelliccia G, Luzi L, Minucci S, Marcaccio M, Pinton P, Rizzuto R, Bernardi P, Paolucci F, Pelicci PG (2005) Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis. Cell 122:221-33.
• Basso E, Fante L, Fowlkes J, Petronilli V, Forte MA, Bernardi P (2005) Properties of the permeability transition pore in mitochondria devoid of Cyclophilin D. J. Biol. Chem. 280:18558-61.

Selected Seminars

Contact

Paolo Bernardi Venetian Institute of Molecular Medicine Via Orus 2 35129 Padua — Italy

Last updated: 28/03/2010, PB ·